Lp-PLA2, also known as platelet activating factor acetylhydrolase (PAF-AH), is encoded by the PLA2G7 gene and is a member of the class PLA2 of the phospholipase family. It is a serine-dependent phospholipase which has a catalytic activity with no need for Ca2+. Human plasma Lp-PLA2 has a molecular weight of 45 kDa and is mainly produced by secretion of macrophages, monocytes, T lymphocytes, mast cells, hepatocytes, and the like, with regulation by inflammatory mediators. For example, its secretion can be inhibited by gamma interferon and lipopolysaccharide but enhanced by platelet activating factor. The main roles of Lp-PLA2 include producing dodecanoic acid inflammatory substances, participating in phospholipid reconstitution and stable equilibrium of biological membrane, lipoprotein metabolism, cell signaling, and host reaction, and facilitating autologous disappearing of necrotic tissue of the body.
Recent studies have shown that Lp-PLA2 can promote the formation of atherosclerotic (AS) plaques and bind to low density lipoprotein (LDL) in the circulation. The current study suggests that coronary heart disease (CHD) is a chronic inflammatory disease, with inflammation involved in various stages of CHD pathogenesis. Lp-PLA2 is secreted by inflammatory cells in the atherosclerotic plaques and is significantly increased in severe atherosclerotic plaques. Therefore, it can be used as a marker for predicting future cardiovascular events of CHD. Other studies have found that Lp-PLA2 is correlated with a risk of congestive heart failure (CHF). The onset of cerebral infarction is closely related to the instability of the AS plaque, while instable plaques, in turn, are prone to embolization or thrombosis, leading to infarction. Lp-PLA2 may be an independent predictor of ischemic stroke, and determination of Lp-PLA2 level may provide predictive information in addition to conventional risk factor assessment, providing insights for guiding the prevention strategy. Studies have found that low-level inflammatory response plays an important role when metabolic syndrome (MS) occurs, wherein the MS health response becomes more obvious with increased activity of Lp-PLA2.
At present, methods for determination of Lp-PLA2 available on the market mainly involve spectrophotometry, latex-enhanced turbidimetry, radioactive immunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA).
Among them, there are currently two types of spectrophotometrical methods for detecting Lp-PLA2. One method uses PAF thioesters as the substrate. Its mechanism involves enzymatic hydrolysis of the thioester group in the sn-2 position on the backbone of the PAF thioester analogue, releasing free thiol, followed by addition of 5'S-disulfide (dinitrobenzoic acid) of the thiol which can be detected, and detection of variation in absorbance at 405 nm. This type of substrates includes 1-thiodecanoyl-2-decanoyl-PC, 1-hexadecanoyl-2-thiohexadecanoyl-PC, and the like. The other method uses PAF analogues with a 4-nitrophenol group. This type of substrate has similar structure with the PAF thioester, except that the thioester group in the sn-2 position is displaced by the 4-nitrophenol group. Its mechanism involves enzymatic hydrolysis of the substrate, releasing the substance with 4-nitrophenol group which has instable properties and immediately decomposes into 4-nitrophenol, and detection of variation in absorbance at 405 nm due to this change, thereby determining the enzyme activity.
In latex-enhanced turbidimetry, Lp-PLA2 in a sample undergoes an antigen-antibody reaction with sensitized latex particles of mouse anti-human Lp-PLA2 antibody in the reagent in a phosphate buffer system, causing agglutination under the action of polyethylene glycol as an accelerator to produce an increased turbidity, and variation in absorbance of the reaction solution is detected at 546 nm wavelength, which is proportional to the Lp-PLA2 content in the sample.
Radioimmunoassay involves a procedure in accordance with the principle of competition mechanism, wherein the binding amount of 1251-Lp-PLA2 with antibody is a function of the Lp-PLA2 content in the standard or sample. The Lp-PLA2 concentration in the sample was determined by separating the binding fraction (B) from the free fraction (F) using an immune separation agent (PR), determining the radioactive intensity of the binding fraction, and processing the data.
Enzyme-linked immunosorbent assay (ELISA) uses a double antibody sandwich method to determine the level of human Lp-PLA2 in whole blood samples. Purified human Lp-PLA2 antibodies are used to coat a microplate to prepare solid phase antibody. Lp-PLA2 is added to the wells coated on the monoclonal antibody and binds with the anti-Lp-PLA2 antibody labeled with horseradish peroxidase (HRP) to form an antibody-antigen-enzyme-labeled antibody complex. TMB (3, 3″, 5, 5″-tetramethylbenzidine) was added as a substrate after thorough washing for coloration. TMB is converted to blue under the catalysis of HRP enzyme and to a final yellow color under the action of acid. The color tone is positively correlated with the Lp-PLA2 content in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader. The content of human Lp-PLA2 in the sample is calculated using a standard curve. Enzyme-linked immunosorbent assay (ELISA) is a commonly used method for determination of Lp-PLA2. However, there is plenty of interferential components in the whole blood samples for Lp-PLA2 measurement, which affects the accuracy of measurement. Besides, the enzyme immunoluminescence antibody coating for selection is limited to a single form and the binding with Lp-PLA2 is not thorough enough, resulting in low detection sensitivity. Relatively large numbers of uncertain factors in the microplate has caused poor repeatability of the test. In addition, enzyme-linked immunoassay is restricted by its degree of automation, rendering a relatively long reaction time.